Project Summary Mitochondrial dysfunction is implicated in several devastating diseases such as heart failure, ischemia- reperfusion injury, diabetes, neurodegeneration, and cancer. Thus, pharmacological interventions targeting mitochondria could become effective strategies for treating these pathological conditions. However, the development of such therapeutic tools is hindered by our incomplete understanding of the molecular mechanisms underlying major mitochondrial functions, including energy production, control of the pace of aging, and control of cell death. Mitochondrial Ca2+ transport dynamically regulates energy production in the heart depending on the energetic demand. This is accomplished by direct Ca2+ binding and activation of key enzymes involved in energy production. However, excessive mitochondrial Ca2+ influx such as that following Ca2+ overload in heart failure can lead to mitochondrial dysfunction and cardiomyocyte death via activation of the mitochondrial permeability transition pore (PTP). The mitochondrial calcium uniporter (MCU) is the principal channel responsible for mitochondrial Ca2+ uptake when the cytosolic Ca2+ level is elevated. MCU is a complex composed a central ion conduction pore and several regulatory subunits. Despite the clear significance of MCU in mitochondrial and heart physiology, rigorous biophysical analyses using direct patch-clamp recording of MCU currents to study the molecular components of the MCU complex have not been achieved. Here we develop the first heterologous expression system for patch-clamp based structure-function analysis of MCU in the inner mitochondrial membrane (IMM). This system employs both whole-IMM and single-channel modes of the patch-clamp technique to study mutants of the MCU pore and auxiliary subunits to enable a comprehensive study of structural features that mediate MCU-complex selectivity, activation, and physiological regulation. We plan to answer two central questions in the field: 1) How does the MCU pore allow selective one-way Ca2+ transport into mitochondria in the presence of much higher concentrations of monovalent cations? 2) How do the auxiliary subunits control MCU gating to achieve precise control over ATP production without causing excessive mitochondrial depolarization or MPT? Answering these questions will provide an essential framework for the development of pharmacological interventions to control MCU activity and mitochondrial function under physiological and pathophysiological conditions.